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GENE TRANSFER AND GENETIC ENGINEERING: - Transformation - Transduction - Conjugation - Gene Transfer Mechanisms Compared - Plasmids - Genetic Engineering

Gene Transfer

- Offspring

*Lateral (horizontal)
- To others in same generation

Bacterial Gene Transfer and Recombinant DNA Technology:
- Transformation
~ Griffith's Experiment

* Griffith's Experiment:

- Griffith's experiment, conducted in 1928 by Frederick Griffith, was one of the first experiments suggesting that bacteria are capable of transferring genetic information through a process known as transformation.

- Griffith used two strains of pneumococcus (Streptococcus pneumoniae) bacteria which infect mice - a type III-S (smooth) and type II-R (rough) strain.
~ The III-S strain covers itself with a polysaccharide capsule that protects it from the host's immune system, resulting in the death of the host, while the II-R strain doesn't have that protective capsule and is defeated by the host's immune system.
~ A German bacteriologist, Fred Neufeld, had discovered the three pneumococcal types (Types I, II, and III) and discovered the Quellung reaction to identify them in vitro.
~ Until Griffith's experiment, bacteriologists believed that the types were fixed and unchangeable, from one generation to another.

- In this experiment, bacteria from the III-S strain were killed by heat, and their remains were added to II-R strain bacteria.
~ While neither alone harmed the mice, the combination was able to kill its host.
~ Griffith was also able to isolate both live II-R and live III-S strains of pneumococcus from the blood of these dead mice.
~ Griffith concluded that the type II-R had been "transformed" into the lethal III-S strain by a "transforming principle" that was somehow part of the dead III-S strain bacteria.

***Today, we know that the "transforming principle" Griffith observed was the DNA of the III-S strain bacteria.
~ While the bacteria had been killed, the DNA had survived the heating process and was taken up by the II-R strain bacteria.
~ The III-S strain DNA contains the genes that form the protective polysaccharide capsule.
~ Equipped with this gene, the former II-R strain bacteria were now protected from the host's immune system and could kill the host. ~ The exact nature of the transforming principle (DNA) was verified in the experiments done by Avery, McLeod and McCarty and by Hershey and Chase.



- Bacterial transformation may be referred to as a stable genetic change brought about by the uptake of naked DNA (DNA without associated cells or proteins) and competence refers to the state of being able to take up exogenous DNA from the environment.

- Transformation is a form of genetic recombination in which a DNA fragment from a dead, degraded bacterium enters a competent recipient bacterium and is exchanged for a piece of DNA of the recipient.

- Transformation usually involves only homologous recombination, a recombination of homologous DNA sequences having nearly the same nucleotide sequences.

- Typically this involves similar bacterial strains or strains of the same bacterial species.

- A few bacteria, such as Neisseria gonorrhoeae, Neisseria meningitidis, Hemophilus influenzae, Legionella pneomophila, Streptococcus pneumoniae, and Helicobacter pylori tend to be naturally competent and transformable.

- Competent bacteria are able to bind much more DNA than noncompetent bacteria.

- Some of these genera also undergo autolysis that then provides DNA for homologous recombination. In addition, some competent bacteria kill noncompetent ones to release DNA for transformation.

- During transformation, DNA fragments (usually about 10 genes long) are released from a dead degraded bacterium and bind to DNA binding proteins on the surface of a competent living recipient bacterium.

- Depending on the bacterium, either both strands of DNA penetrate the recipient, or a nuclease degrades one strand of the fragment and the remaining DNA strand enters the recipient. This DNA fragment from the donor is then exchanged for a piece of the recipient's DNA by means of RecA proteins. This involves breakage and reunion of paired DNA segments

- Mechanism

*Transformation Mechanism:

- During transformation, DNA fragments (usually about 10 genes long) are released from a dead degraded bacterium and bind to DNA binding proteins on the surface of a competent living recipient bacterium.

- Depending on the bacterium, either both strands of DNA penetrate the recipient, or a nuclease degrades one strand of the fragment and the remaining DNA strand enters the recipient.

- This DNA fragment from the donor is then exchanged for a piece of the recipient's DNA by means of RecA proteins.

- This involves breakage and reunion of paired DNA segments

*Transformation Mechanism in Gene Recombination:

*Transformation Mechanism in Gene Recombination:

- A number of donor cells break apart and an explosive release and fragmentation of DNA follows.
~ A segment of double stranded DNA containing about 10-20 genes then passes through the cell wall and membrane of a recipient cell.
~ Only a few competent recipient cells can take up the DNA.
~ After entry into cell, an enzyme dissolves one strand of DNA leaving the second strand to be incorporated.

- This strand then displaces a segment from a strand of the recipient's DNA.
~ The displaced DNA is dissolved by another enzyme in the cell.
~ The cell is now transformed. It will display its own traits as well as those coded by the new DNA.

- Transformation may also take place by the incorporation of plasmids to competent cells.
~ In this case, no DNA is displaced.
~ Rather, the plasmid adds genes to those already in the cell and multiplies along with the cell.

- Competence Factor
~ Natural Competance
~ Artificial Competance

*Competence Factor:

- In microbiology, genetics, cell biology and molecular biology, competence is the ability of a cell to take up extracellular ("naked") DNA from its environment.
~ Competence may be differentiated between natural competence, a genetically specified ability of bacteria which is thought to occur under natural conditions as well as in the laboratory.
~ And induced or artificial competence, which arises when cells in laboratory cultures are treated to make them transiently permeable to DNA.

- Natural Competence

*Natural Competence:

- About 1% of bacterial species are capable of naturally taking up DNA under laboratory conditions; more may be able to take it up in their natural environments.
~ DNA material can be transferred between different strains of bacteria, in a process that is called horizontal gene transfer.
~ Some species upon cell death release their DNA to be taken up by other cells, however transformation works best with DNA from closely related species.
~ These naturally competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s).
~ The transport of the exogeneous DNA into the cells may require proteins that are involved in the assembly of type IV pili and type II secretion system, as well as DNA translocase complex at the cytoplasmic membrane.

- Natural Competence Mechanism:

- Due to the differences in structure of the cell envelop between Gram-positive and Gram-negative bacteria, there are some some differences in the mechanisms of DNA uptake in these cells, however most of them share common features that involve related proteins.

- The DNA first binds to the surface of the competent cells on a DNA receptor, and passes through the cytoplasmic membrane via DNA translocase.

- Only single-stranded DNA may pass through, one strand is therefore degraded by nucleases in the process, and the translocated single-stranded DNA may then be integrated into the bacterial chromosomes by a RecA-dependent process.

- In Gram-negitive cells, due to the presence of an extra membrane, the DNA requires the presence of a channel formed by secretins on the outer membrane.

- Pilin may be required for competence however its role is uncertain.

- The uptake of DNA is generally non-sequence specific, although in some species the presence of specific DNA uptake sequences may facilitate efficient DNA uptake.

- Artificial Competence

*Artificial competence :

- Artificial competence can be induced in laboratory procedures that involves making the cell passively permeable to DNA by exposing it to conditions that do not normally occur in nature.
~ Typically the cells are incubated in a solution containing divalent cations, most commonly calcium chloride solution under cold condition, and then exposed to a pulse of heat shock.
~ However, the mechanism of the uptake of DNA via chemically-induced competence in this calcium chloride transformation is unclear. The surface of bacteria such as E. coli is negatively charged due to phospholipids and lipopolysaccharides on its cell surface, and the DNA is also negatively-charged.
~ One function of the divalent cation therefore would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface.
~ It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure of the cells making it more permeable to DNA.
~ The heat-pulse is thought to create a thermal imbalance on either side of the cell membrane, which forces the DNA to enter the cells either through cell pores or the damaged cell wall.

- Electroporation


- Electroporation is another method of promoting competence.
~ In this method the cells are briefly shocked with an electric field of 10-20 kV/cm which is thought to create holes in the cell membrane through which the plasmid DNA may enter.
~ After the electric shock the holes are rapidly closed by the cell's membrane-repair mechanisms.

- Mechanism for Insertion

*Mechanism for Insertion

A number of mechanisms are available to transfer DNA into plant cells:

~ Agrobacterium mediated transformation is the easiest and most simple plant transformation. Plant tissue (often leaves) are cut into small pieces, e.g. 10x10mm, and soaked for 10 minutes in a fluid containing suspended Agrobacterium. Some cells along the cut will be transformed by the bacterium, that inserts its DNA into the cell. Placed on selectable rooting and shooting media, the plants will regrow. Some plants species can be transformed just by dipping the flowers into suspension of Agrobacterium and then planting the seeds in a selective medium. Unfortunately, many plants are not transformable by this method.
~ Particle bombardment: Particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. This method also allows transformation of plant plastids. The transformation efficiency is lower than in Agrobacterium mediated transformation, but most plants can be transformed with this method.
~ Electroporation: make transient holes in cell membranes using electric shock; this allows DNA to enter as described above for Bacteria.
~ Viral transformation (transduction): Package the desired genetic material into a suitable plant virus and allow this modified virus to infect the plant. If the genetic material is DNA, it can recombine with the chromosomes to produce transformant cells. However genomes of most plant viruses consist of single stranded RNA which replicates in the cytoplasm of infected cell. For such genomes this method is a form of transfection and not a real transformation, since the inserted genes never reach the nucleus of the cell and do not integrate into the host genome. The progeny of the infected plants is virus free and also free of the inserted gene.

- Animals:

~ Tranfsection- Introduction of DNA into animal cells is usually called transfection.

- Significance

*Significance of Transformation:

- Since the 1940's, transformation has also been demonstrated in species of Neisseria, Bacillus, Haemophilus, Azotohacter and Streptococcus.
~ The process involves the transfer of DNA from the fragments of donor cells into the cytoplasm of a live recipient cell.
~ Sections of single stranded or double stranded DNA may be taken up but only a single strand will align with the bacterial chromosome and becomes incorporated into it.

- Transformations in bacteria have been observed in the ability to form a capsule, a drug resistance and pathogenicity, and in nutritional patterns.
~ Transformations are not common, however, because the large fragments of DNA molecule can not pass through the recipient's cell wall or membrance.
~ In nature, transformation may increase the pathogenicity of an organism.

Transduction :

*Transduction :

-Transduction is the process by which DNA is transferred from one bacterium to another by a virus.
~ It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector.
~ Transduction does not require cell-to-cell contact (which occurs in conjugation), and it is DNAase resistant (transformation is susceptable to DNAase).
~ Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell's genome.

- When bacteriophages (viruses that infect bacteria) infect a bacterial cell, their normal mode of reproduction is to harness the replicational, transcriptional, and translation machinery of the host bacterial cell to make numerous virions, or complete viral particles, including the viral DNA or RNA and the protein coat.

- Discovery

*Discovery of Transduction:

-Transduction was discovered by Norton Zinder and Joshua Lederberg at the University of Wisconsin-Madison in 1951.
~ However, the mechanism of general transduction as well as specialized transduction was discovered a few years later, by Esther Lederberg;

- Mechanisms of Transduction- Bacteriophages
~Lytic and Lysogenic (temperate) Cycles

*Lytic and Lysogenic (temperate) Cycles:
- Transduction happens through either the lytic cycle or the lysogenic cycle.

- If the lysogenic cycle is adopted, the phage chromosome is integrated (by covalent bonds) into the bacterial chromosome, where it can remain dormant for thousands of generations.
- If the lysogen is induced (by UV light for example), the phage genome is excised from the bacterial chromosome and initiates the lytic cycle, which culminates in lysis of the cell and the release of phage particles.
- The lytic cycle leads to the production of new phage particles which are released by lysis of the host.

- Generalized Transduction

*Generalized Transduction:

- Generalized transduction is the process by which any bacterial gene may be transferred to another bacterium via a bacteriophage, and typically carries only bacterial DNA and no viral DNA.
~ In essence, this is the packaging of bacterial DNA into a viral envelope. This may occur in two main ways, recombination and headful packaging.

- If bacteriophages undertake the lytic cycle of infection upon entering a bacterium, the virus will take control of the cell's machinery for use in replicating its own viral DNA. If by chance bacterial chromosomal DNA is inserted into the viral capsid which is usually used to encapsulate the viral DNA, the mistake will lead to generalized transduction.

- If the virus replicates using 'headful packaging', it attempts to fill the nucleocapsid with genetic material. If the viral genome results in spare capacity, viral packaging mechanisms may incorporate bacterial genetic material into the new virion.

- The new virus capsule now loaded with part bacterial DNA continues to infect another bacterial cell.
~ This bacterial material may become recombined into another bacterium upon infection.

- When the new DNA is inserted into this recipient cell it can fall to one of three fates:

1. The DNA will be absorbed by the cell and be recycled for spare parts.
2. If the DNA was originally a plasmid, it will re-circularize inside the new cell and become a plasmid again.
3. If the new DNA matches with a homologous region of the recipient cell's chromosome, it will exchange DNA material similar to the actions in conjugation.

***This type of recombination is random and the amount recombined depends on the size of the virus being used.

- Specialized Transduction

*Specialized Transduction:

-Specialized transduction is the process by which genes that are near the bacteriophage genome may be transferred to another bacterium via a bacteriophage.
~ The genes that get transferred (donor genes) always depend on where the phage genome is located on the chromosome.
~ This second type of recombination event which is the result of mistakes in the transition from a virus' lysogenic to lytic cycle is called specialized transduction, and non-viral DNA is carried as an insertion/substitution.
~ If a virus incorrectly removes itself from the bacterial chromosome, bacterial DNA from either end of the phage DNA may be packaged into the viral capsid.
~Specialized transduction leads to three possible outcomes:

1. DNA can be absorbed and recycled for spare parts.
2. The bacterial DNA can match up with a homologous DNA in the recipient cell and exchange it. The recipient cell now has DNA from both itself and the other bacterial cell.
3. DNA can insert itself into the genome of the recipient cell as if still acting like a virus resulting in a double copy of the bacterial genes.

- When the partially encapsulated phage material infects another cell and becomes a "prophage" (is covalently bonded into the infected cell's chromosome), the partially coded prophage DNA is called a "heterogenote".

- Esther Lederberg, Larry Morse, Herman Kalckar, Michael Yarmolinsky, and Yukinori Hirota went on to do detailed studies of Galactosemia.
~ Specialized transduction was used in these studies for gene mapping.
~ At about this time, Esther Lederberg, Julius Adler, and Enrico Calef were also engaged in similar research involving Maltophilia.

***Example of specialized transduction is λ phages in Escherichia coli discovered by Esther Lederberg as well as Fertility Factor F, also discovered by Esther Lederberg.

- Significance

***Transduction Significance-
- Can be used in genetic mapping.

- Transduction and specialized transduction is especially important because they explain how anti-biotic drugs become ineffective due to the transfer of resistant genes between bacteria.
~ In addition, hopes to create medical methods of genetic modification of diseases such as Duschenne/Becker Muscular Dystrophy are based upon these methodologies.



- Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells.

- Discovered in 1946 by Joshua Lederberg and Edward Tatum, conjugation is a mechanism of horizontal gene transfer as are transformation and transduction although these two other mechanisms do not involve cell-to-cell contact.

- Bacterial conjugation is often incorrectly regarded as the bacterial equivalent of sexual reproduction or mating since it involves the exchange of genetic material.
~ During conjugation the donor cell provides a conjugative or mobilizable genetic element that is most often a plasmid or transposon.
~ Most conjugative plasmids have systems ensuring that the recipient cell does not already contain a similar element.

- The genetic information transferred is often beneficial to the recipient.
~ Benefits may include antibiotic resistance, xenobiotic tolerance or the ability to use new metabolites.
~ Such beneficial plasmids may be considered bacterial endosymbionts.
~ Other elements, however, may be viewed as bacterial parasites and conjugation as a mechanism evolved by them to allow for their spread.

- Mechanism

*Conjugation Mechanism:

1- Donor cell produces pilus.
2- Pilus attaches to recipient cell and brings the two cells together.
3- The mobile plasmid is nicked and a single strand of DNA is then transferred to the recipient cell.
4- Both cells synthesize a complementary strand to produce a double stranded circular plasmid and also reproduce pili; both cells are now viable donors.

- Significance

***Significance- Also used for mapping.

- Conjugation is a convenient means for transferring genetic material to a variety of targets.

- In laboratories successful transfers have been reported from bacteria to yeast, plants, mammalian cells and isolated mammalian mitochondria.

- Conjugation has advantages over other forms of genetic transfer including minimal disruption of the target's cellular envelope and the ability to transfer relatively large amounts of genetic material .

- In plant engineering, Agrobacterium-like conjugation complements other standard vehicles such as tobacco mosaic virus (TMV).

- While TMV is capable of infecting many plant families these are primarily herbaceous dicots.

- Agrobacterium-like conjugation is also primarily used for dicots, but monocot recipients are not uncommon.

- Transfer of F plasmids
a. F+
b. F
c. F pilus
d. Conjugation bridge

*Bacterial Conjugation

- The transfer of genetic information via direct cell-cell contact;
~ this process is mediated by fertility factors (F plasmids)

- F+ ¥ F- mating -
~ In E. coli and other gram-negative bacteria, an F plasmid moves from the donor (F+) to a recipient (F-) while being replicated by the rolling circle mechanism
~ The displaced strand is transferred via a sex pilus and then copied to produce double-stranded DNA;
~ The donor retains the other parental DNA strand and its complement; thus the recipient becomes F+ and the donor remains F+
~ Chromosomal genes are not transferred
~ In gram-positive bacteria, the sex pilus is not necessarily required for transmission; generally fewer genes are transferred

- Hfr conjugation
~ F plasmid integration into the host chromosome results in an Hfr strain of bacteria
~ The mechanics of conjugation of Hfr strains are similar to those of F+ strains
~ The initial break for rolling-circle replication is at the integrated plasmidís origin of transfer site
~ Part of the plasmid is transferred first
~ Chromosomal genes are transferred next
~ The rest of the plasmid is transferred last

- Complete transfer of the chromosome takes approximately 100 minutes, but the conjugation bridge does not usually last that long;
~ therefore, the entire F factor is not usually transferred, and the recipient remains F-

- F¢ conjugation (sexduction)
~ When an integrated F plasmid leaves the chromosome incorrectly, it may take with it some chromosomal genes from one side of the integration site;
~ this results in the formation of an abnormal plasmid called an F¢ plasmid
~ The F¢ cell (cell harboring an F¢ plasmid) retains its genes, although some of them are in the chromosome and some are on the plasmid;
~ in conjugation, an F¢ cell behaves as an F+ cell, mating only with F- cells
~ The chromosomal genes included in the plasmid are transferred with the rest of the plasmid, but other chromosomal genes usually are not
~ The recipient becomes an F¢ cell, and a partially diploid merozygote



- In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA.
~ They are double-stranded and, in many cases, circular.
~ Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre ring in Saccharomyces cerevisiae).

- Plasmid sizes vary from 1 to over 1,000 kbp.
~ The number of identical plasmids in a single cell can range anywhere from one to even thousands under some circumstances.
~ Plasmids can be considered part of the mobilome because they are often associated with conjugation, a mechanism of horizontal gene transfer.

- The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952.

- Plasmids are considered "replicons", capable of autonomous replication within a suitable host.
~ Plasmids can be found in all three major domains: Archea, Bacteria, and Eukarya.
~ Similar to viruses, plasmids are not considered by some to be a form of "life".
~ Unlike viruses, plasmids are "naked" DNA and do not encode genes necessary to encase the genetic material for transfer to a new host, though some classes of plasmids encode the sex pilus necessary for their own transfer.
~ Plasmid host-to-host transfer requires direct, mechanical transfer by conjugation or changes in host gene expression allowing the intentional uptake of the genetic element by transformation.
~ Microbial transformation with plasmid DNA is neither parasitic nor symbiotic in nature, because each implies the presence of an independent species living in a commensal or detrimental state with the host organism. ~ Rather, plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state.
~ Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or the proteins produced may act as toxins under similar circumstances.
~ Plasmids can also provide bacteria with the ability to fix elemental nitrogen or to degrade recalcitrant organic compounds that provide an advantage when nutrients are scarce.

-Resistance Transfer Factor

*Resistance Transfer Factor RTF - Bacteriocin Plasmids:

- The discovery of RTF was first made in Japan during an outbreak of bacterial dysentery when a strain of Shiegella dysentriae was found to be simultaneously resistant to sulphonamides, streptomycin, chloram phenicol and tetracycline.
~ Subsequently it was found that this simultaneous resistance to four drugs could also be lost by a single mutation.

- Since then, multiple drug resistant bacteria have been isolated from every part of the world and it know well established that this property is controlled by the RTF.
~ The number of inhibitory substances for which resistance may be mediated by R-factors has now grown to ten or more antibiotics and several heavy metals such as Hg, Cd, Ni, and Co.
~ It has also been found that different R factors have different combinations or resistance genes ranging from 1-8.

- The RTF consists of two components, one called the RTF.(resistance transfer factor) carrying genes for replication and transmission of the plasmid and the second consisting of one or more sequentially linked R determinants (resistance determinants).
~ The RTF region of several R-factors has been characterized and found to have molecular weights as large as 60 million daltons while the R-determinants have molecular weights of about ten million.

- These two segments of the RTF have been found to have different G+C content.
~ Several R-factors are also capable of dissociating into independently replicating RTFs and R-determinants.
~ The latter, appear to be replicons but arc incapable of being transferred unless they are associated with the RTF.
~The stability of the association has been found to vary with the R-factor and the host.
~Some R-factors are stable in one host but dissociate readily in another.

- In nature, bacteria may contain either the R-determinants or only the RTF.
~ Such strains can be detected by simple techniques which Anderson and his coworkers used to find Salmonella strains carrying RTF.
~ In this a tester strain was produced by early interruption of mating between a R-factor donor which contained an R-determinant but not RTF.
~ This tester was then mated with a potential RTF donor or with a drug sensitive recipient.

- The transfer of an RTF from the donor was detected by the acquired ability of the tester to transfer its drug resistance to another recipient.
~ Some R-determinants are capable of autonomous replication but not of self transfer.
~ The genes controlling the conjugation process are located only on RTF since only RTF carries genes for replication and transmission, of the plasmid.

- Plasmids isolated from bacteria fall into a number of compatibility groups.
~ A compatibility group is defined as a group of plasmids which can stably establish in the same cell and is not related to each other.
~ That is, only plasmids representing different compatibility groups can co exist and replicate side by side in the same host.
~ This phenomenon of incompatibility reflects competition between members of a given group for common host cell functions for replication.

- As a Consequence even if a cell has two incompatible plasmids to begin with later as replication proceeds only one type is selected and established.
~ Compatibility and incompatibility is therefore, more of a reflection of interaction between the plasmids and the cell.
~ Naturally occurring bacteria can have as many as 7-8 different plasmids

- Resistance genes (R genes)
- How are antibiotic resistance genes on some plasmids used in genetic engineering?

*Resistance genes (R genes):

Schematic representation of how antibiotic resistance evolves via natural selection. The top section represents a population of bacteria before exposure to an antibiotic. The middle section shows the population directly after exposure, the phase in which selection took place. The last section shows the distribution of resistance in a new generation of bacteria. The legend indicates the resistance levels of individuals.

- Diagram depicting antibiotic resistance through alteration of the antibiotic's target site, modeled after MRSA's resistance to penicillin. Beta-lactam antibiotics permanently inactivate PBP enzymes, which are essential for bacterial life, by permanently binding to their active sites. MRSA, however, expresses a PBP that will not allow the antibiotic into its active site.

Antibiotic resistance can be a result of horizontal gene transfer,[36] and also of unlinked point mutations in the pathogen genome at a rate of about 1 in 108 per chromosomal replication. The antibiotic action against the pathogen can be seen as an environmental pressure; those bacteria which have a mutation allowing them to survive will live on to reproduce. They will then pass this trait to their offspring, which will result in the evolution of a fully resistant colony.

The four main mechanisms by which microorganisms exhibit resistance to antimicrobials are:

1.Drug inactivation or modification: for example, enzymatic deactivation of Penicillin G in some penicillin-resistant bacteria through the production of β-lactamases.
2.Alteration of target site: for example, alteration of PBP—the binding target site of penicillins—in MRSA and other penicillin-resistant bacteria.
3.Alteration of metabolic pathway: for example, some sulfonamide-resistant bacteria do not require para-aminobenzoic acid (PABA), an important precursor for the synthesis of folic acid and nucleic acids in bacteria inhibited by sulfonamides. Instead, like mammalian cells, they turn to utilizing preformed folic acid.
4.Reduced drug accumulation: by decreasing drug permeability and/or increasing active efflux (pumping out) of the drugs across the cell surface.

- There are three known mechanisms of fluoroquinolone resistance. Some types of efflux pumps can act to decrease intracellular quinolone concentration.[38] In gram-negative bacteria, plasmid-mediated resistance genes produce proteins that can bind to DNA gyrase, protecting it from the action of quinolones. Finally, mutations at key sites in DNA gyrase or Topoisomerase IV can decrease their binding affinity to quinolones, decreasing the drug's effectiveness.[39] Research has shown that the bacterial protein LexA may play a key role in the acquisition of bacterial mutations giving resistance to quinolones and rifampicin.

- Antibiotic resistance can also be introduced artificially into a microorganism through laboratory protocols, sometimes used as a selectable marker to examine the mechanisms of gene transfer or to identify individuals that absorbed a piece of DNA that included the resistance gene and another gene of interest. A recent study demonstrates the extent of horizontal gene transfer among Staphylococcus to be much greater than one previously expected, and encompasses genes with functions beyond antibiotic resistance and virulence, and beyond genes residing within the mobile genetic elements.

***Antibiotic resistance is an important tool for genetic engineering. By constructing a plasmid which contains an antibiotic resistance gene as well as the gene being engineered or expressed, a researcher can ensure that when bacteria replicate, only the copies which carry along the plasmid survive. This ensures that the gene being manipulated passes along when the bacteria replicates.

- The most commonly used antibiotics in genetic engineering are generally "older" antibiotics which have largely fallen out of use in clinical practice. These include:

- ampicillin
- kanamycin
- tetracycline
- chloramphenicol

- Industrially the use of antibiotic resistance is disfavored since maintaining bacterial cultures would require feeding them large quantities of antibiotics. Instead, the use of auxotrophic bacterial strains (and function-replacement plasmids) is preferred.

- Displacins


- How can resistance plasmids be removed from a bacterium?
By displacins.

- What are displacins?
Pieces of DNA isolated from soil bacteria and attached to usually harmless E.coli bacteria.

- What do displacins do?
Move from the E. coli into the pathogens where they literally displace plasmids carrying harmful genes, leaving the former pathogenes now harmless.

- Transposons

are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's genome size. Barbara McClintock's discovery of these jumping genes early in her career earned her a Nobel prize in 1983.

- Transposons make up a large fraction of the C-value of eukaryotic cells. Transposons are often considered "junk DNA". In Oxytricha, which has a unique genetic system, they play a critical role in its development. Transposons are very useful to researchers as a means to alter DNA inside a living organism.

- The first transposon was discovered in the plant maize (Zea mays, corn species), and is named dissociator (Ds). Likewise, the first transposon to be molecularly isolated was from a plant (Snapdragon). Appropriately, transposons have been an especially useful tool in plant molecular biology. Researchers use transposons as a means of mutagenesis. In this context, a transposon jumps into a gene and produces a mutation. The presence of the transposon provides a straightforward means of identifying the mutant allele, relative to chemical mutagenesis methods.

- Sometimes the insertion of a transposon into a gene can disrupt that gene's function in a reversible manner, in a process called insertional mutagenesis; transposase-mediated excision of the transposon restores gene function. This produces plants in which neighboring cells have different genotypes. This feature allows researchers to distinguish between genes that must be present inside of a cell in order to function (cell-autonomous) and genes that produce observable effects in cells other than those where the gene is expressed.

- Transposons are also a widely used tool for mutagenesis of most experimentally tractable organisms. The Sleeping Beauty transposon system has been used extensively as an insertional tag for identifying cancer genes .

- The Tc1/mariner-class transposon Sleeping Beauty transposon system, awarded as the Molecule of the Year 2009 is active in mammalian cells and are being investigated for use in human gene therapy

- Transposons are mutagens. They can damage the genome of their host cell in different ways [14]:

A transposon or a retroposon that inserts itself into a functional gene will most likely disable that gene.
After a transposon leaves a gene, the resulting gap will probably not be repaired correctly.
Multiple copies of the same sequence, such as Alu sequences can hinder precise chromosomal pairing during mitosis and meiosis, resulting in unequal crossovers, one of the main reasons for chromosome duplication.
Diseases that are often caused by transposons include hemophilia A and B, severe combined immunodeficiency, porphyria, predisposition to cancer, and Duchenne muscular dystrophy.[15][16]

Additionally, many transposons contain promoters which drive transcription of their own transposase. These promoters can cause aberrant expression of linked genes, causing disease or mutant phenotypes.

- Bacteriocinogens


- Medical significance
Bacteriocins are of interest in medicine because they are made by non-pathogenic bacteria that normally colonize the human body. Loss of these harmless bacteria following antibiotic use may allow opportunistic pathogenic bacteria to invade the human body.

- Bacteriocins have also been suggested as a cancer treatment. They have shown distinct promise as a diagnostic agent for some cancers, but their status as a form of therapy remains experimental and outside the main thread of cancer research. Partly this is due to questions about their mechanism of action and the presumption that anti-bacterial agents have no obvious connection to killing mammalian tumor cells. Some of these questions have been addressed, at least in part.

Bacteriocins (which?) were tested as AIDS drugs (around 1990) but not progressed beyond in-vitro tests on cell lines.

Genetic Engineering

*Genetic engineering,
- also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest. The introduction of new DNA does not require the use of classical genetic methods, however traditional breeding methods are typically used for the propagation of recombinant organisms.

An organism that is generated through the introduction of recombinant DNA is considered to be a genetically modified organism. The first organisms genetically engineered were bacteria in 1973 and then mice in 1974. Insulin-producing bacteria were commercialized in 1982 and genetically modified food has been sold since 1994.

The most common form of genetic engineering involves the insertion of new genetic material at an unspecified location in the host genome. This is accomplished by isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence containing the required genetic elements for expression, and then inserting this construct into the host organism. Other forms of genetic engineering include gene targeting and knocking out specific genes via engineered nucleases such as zinc finger nucleases or engineered homing endonucleases.

Genetic engineering techniques have been applied in numerous fields including research, biotechnology, and medicine. Medicines such as insulin and human growth hormone are now produced in bacteria, experimental mice such as the oncomouse and the knockout mouse are being used for research purposes and insect resistant and/or herbicide tolerant crops have been commercialized. Genetically engineered plants and animals capable of producing biotechnology drugs more cheaply than current methods (called pharming) are also being developed and in 2009 the FDA approved the sale of the pharmaceutical protein antithrombin produced in the milk of genetically engineered goats.

Genetic Engineering:
- Definition


-Genetic engineering alters the genetic makeup of an organism using techniques that introduce heritable material prepared outside the organism either directly into the host or into a cell that is then fused or hybridized with the host. This involves using recombinant nucleic acid (DNA or RNA) techniques to form new combinations of heritable genetic material followed by the incorporation of that material either indirectly through a vector system or directly through micro-injection, macro-injection and micro-encapsulation techniques. Genetic engineering does not include traditional animal and plant breeding, in vitro fertilisation, induction of polyploidy, mutagenesis and cell fusion techniques that do not use recombinant nucleic acids or a genetically modified organism in the process. Cloning and stem cell research, although not considered genetic engineering, are closely related and genetic engineering can be used within them. Synthetic biology is an emerging discipline that takes genetic engineering a step further by introducing artificially synthesized genetic material from raw materials into an organism.

If genetic material from another species is added to the host, the resulting organism is called transgenic. If genetic material from the same species or a species that can naturally breed with the host is used the resulting organism is called cisgenic. Genetic engineering can also be used to remove genetic material from the target organism, creating a knock out organism.[6] In Europe genetic modification is synonymous with genetic engineering while within the United States of America it can also refer to conventional breeding methods. Within the scientific community, the term genetic engineering is not commonly used; more specific terms such as transgenic are preferred.

Genetic Engineering:
- Genetic Fusion

*Genetic Fusion:

-The accidental joining of DNA of two genes, such as can occur in a translocation or inversion. Gene fusions can give rise to hybrid proteins or to the misregulation of the transcription of one gene by the cis regulatory elements (enhancers) of another.

- Genetic Fusion allows transposition of genes from one location on a chromosome to another (also could involve deletion of DNA sequence - if gal and bio operon are adjacent and we delete the control genes of the bio operon, the coupling of the 2 operons would be genetic fusion. Gal genes would now control the entire operon)

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